Two-Dimensional Separation Using High-pH and Low-pH Reversed Phase Liquid Chromatography for Top-down Proteomics

Abstract

Advancements in chromatographic separation are critical to in-depth top-down proteomics of complex intact protein samples. Reversed-phase liquid chromatography is the most prevalent technique for top-down proteomics. However, in cases of high complexities and large dynamic ranges, 1D-RPLC may not provide sufficient coverage of the proteome. To address these challenges, orthogonal separation techniques are often combined to improve the coverage and the dynamic range of detection. In this study, a "salt-free" high-pH RPLC was evaluated as an orthogonal dimension of separation to conventional low-pH RPLC with top-down MS. The RPLC separations with low-pH conditions (pH=2) and high-pH conditions (pH=10) were compared to confirm the good orthogonality between high-pH and low-pH RPLC's. The offline 2D RPLC-RPLC-MS/MS analyses of intact E. coli samples were evaluated for the improvement of intact protein identifications as well as intact proteoform characterizations. Compared to the 163 proteins and 328 proteoforms identified using a 1D RPLC-MS approach, 365 proteins and 886 proteoforms were identified using the 2D RPLC-RPLC top-down MS approach. Our results demonstrate that the 2D RPLC-RPLC top-down approach holds great potential for in-depth top-down proteomics studies by utilizing the high resolving power of RPLC separations and by using mass spectrometry compatible buffers for easy sample handling for online MS analysis.

Keywords: 2D separation; Top-down proteomics; proteoform.

Figure 1

The separation of three standard proteins using (A) low pH RPLC, and (B) high pH RPLC. The retention time was normalized to the percentage of mobile phase B.

Figure 2

Comparison of low-pH RPLC and high-pH RPLC as the first-dimension separation. (A) UV chromatograms SDS-PAGE using low-pH RPLC; (B) UV chromatogram and SDS-PAGE using high-pH RPLC. (C) Venn diagram of unique proteins identified using bottom-up proteomics. (D) Histogram of the fraction shift from low pH RPLC to high pH RPLC.

Figure 3

Venn diagram of numbers of (A) proteins and (B) proteoforms identified using 1D and 2D methods. (C) Base peak chromatograms of 4 representative fractions.

Figure 4

2D pH RP/RPLC/MS analysis on E.coli proteins. (A) Base peak chromatogram of second dimension top-down analysis of fraction 19. (B) Representative mass spectra of three proteins identified in fraction 19. The three proteins are periplasmic protein, peptidyl-prolyl cis-trans isomerase A, and mono-oxygenase, subunit of predicted monooxygenase. (C) Overlay of observed isotopic distrubution and theoretical isotopic distribution (red cycles).

Figure 5

Five-minute segment (RT = 90 min to 95 min) from LC/MS runs of 1D and 2D methods.

Figure 6

Different proteoforms of ref|YP_002998928.1 apo-acyl carrier protein (gi|251784624) identified using 1D and 2D methods.